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US4172765A
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Method for the quantitative determination of alpha-amylase
Assignee: Bayer Corp
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Filing: 1977-09-14
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Granted: 1979-10-30
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Expires: 1997-09-14
Abstract
Abstract This invention relates to a method and apparatus for rapid quantitative determination of alpha -amylase in aqueous samples such as blood serum, etc. The method comprises a flow-through of the sample through various immobilized reagents contained in sequential stages. A scavenger stage initially removes glucose originally present in the sample and comprises immobilized glucose oxidase and catalase. The glucose-free sample flows through an immobilized starch stage substrate preferably containing a high percentage of amylose to quantitatively react with the alpha -amylase in the sample and produce oligosaccharides. The sample with the oligosaccharides flows through a glucose-generating stage containing immobilized glucoamylase which converts the oligosaccharides to glucose. The glucose-containing sample enters a detection stage wherein the glucose is converted to H2O2 by flowing through immobilized glucose oxidase, and the H2O2 produced is quantitatively detected in detection means such as a polarographic cell. The immobilized reagents are preferably contained in an apparatus which comprises at least one column in combination with the detection means and a readout means.
Inventors
- 👤 Melvin H. Keyes
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-26 21:24
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/34; C12Q1/40; C12M; C12M21/00; C12M21/18; C12M23/00; C12M23/58; C12M41/00; C12M41/30; C12M41/32; Y; Y10; Y10S; Y10S435/00; Y10S435/817
Claims Analysis
Total Claims: 44
Claim 1
Independent Claim
156 words
A method for the quantitative determination of the pancreatic and salivary α-amylases in an aqueous solution sample containing glucose as a contaminant comprising the steps of passing said sample through a scavenger stage containing immobilized scavenger reagents capable of reacting with and removing said glucose as a sample contaminant in the said determination, passing said glucose-free sample through a substrate stage comprising an immobilized starch reagent capable of reacting quantitatively with said α-amylases to produce oligosaccharides, passing said oligosaccharides-containing sample through a glucose-generating stage comprising an immobilized glucose-generating reagent capable of reacting said oligosaccharides to a glucose reaction product, passing said glucose reaction product containing sample to a detection stage comprising an immobilized reagent capable of reacting with said glucose reaction product to generate hydrogen peroxide, determining the amount of said hydrogen peroxide generated in detection means, and relating the detection reading from said detection means to the quantitative amount of α-amylases contained in said sample.
Claim 2
Independent Claim
156 words
A method for the quantitative determination of the pancreatic and salivary α-amylases in an aqueous solution sample containing glucose as a contaminant comprising the steps of passing said sample through a scavenger stage containing immobilized scavenger reagents capable of reacting with and removing said glucose as a sample contaminant in the said determination, passing said glucose-free sample through a substrate stage comprising an immobilized starch reagent capable of reacting quantitatively with said α-amylases to produce oligosaccharides, passing said oligosaccharides-containing sample through a glucose-generating stage comprising an immobilized glucose-generating reagent capable of reacting said oligosaccharides to a glucose reaction product, passing said glucose reaction product containing sample to a detection stage comprising an immobilized reagent capable of reacting with said glucose reaction product to generate hydrogen peroxide, determining the amount of said hydrogen peroxide generated in detection means, and relating the detection reading from said detection means to the quantitative amount of α-amylases contained in said sample.
Claim 3
Dependent Claim
17 words
The method of claim 1, wherein said aqueous solution sample is diluted with an aqueous buffered diluent.
Claim 4
Dependent Claim
15 words
The method of claim 1, wherein said scavenger reagents comprise coimmobilized glucose oxidase and catalase.
Claim 5
Dependent Claim
19 words
The method of claim 1, wherein said scavenger stage comprises an immobilized catalase reagent subsequent to said scavenger reagents.
Claim 6
Dependent Claim
17 words
The method of claim 1, wherein said immobilized starch reagent is a starch high in amylose content.
Claim 7
Dependent Claim
12 words
The method of claim 1, wherein said starch reagent is potato starch.
Claim 8
Dependent Claim
11 words
The method of claim 1, wherein said glucose-generating reagent is glucoamylase.
Claim 9
Dependent Claim
11 words
The method of claim 1, wherein said glucose-generating reagent is maltase.
Claim 10
Dependent Claim
13 words
The method of claim 1, werein said oligosaccharides contain 2-6 monomers of glucose.
Claim 11
Dependent Claim
20 words
The method of claim 1, wherein said glucose reaction product in said detection stage is reacted with immobilized glucose oxidase.
Claim 12
Dependent Claim
13 words
The method of claim 1, wherein said detection stage comprises polarographic cell means.
Claim 13
Dependent Claim
12 words
The method of claim 1, wherein said detection stage comprises spectrographic means.
Claim 14
Dependent Claim
21 words
The method of claim 1, wherein said scavenger reagents comprise coimmobilized glucose oxidase and catalase on a hard inorganic porous support.
Claim 15
Dependent Claim
11 words
The method of claim 13, werein said porous support is glass.
Claim 16
Dependent Claim
19 words
The method of claim 1, wherein said substrate stage comprises an immobilized amylose starch on a porous refractory support.
Claim 17
Dependent Claim
11 words
The method of claim 15, wherein said porous support is alumina.
Claim 18
Dependent Claim
19 words
The method of claim 1, wherein said substrate stage comprises an amylose starch immobilized and cross-linked on porous alumina.
Claim 19
Dependent Claim
11 words
The method of claim 17, wherein said alumina is acid activated.
Claim 20
Dependent Claim
26 words
The method of claim 1, wherein said aqueous sample is buffered with an aqueous buffered diluent to pH in the range of about 5 to 7.
Claim 21
Dependent Claim
44 words
The method of claim 1, wherein said sample containing said glucose reaction product in said detection stage is passed in direct contact with detection means comprising polarographic electrodes and polarographically determining hydrogen peroxide in said reaction mixture while in direct contact with said electrodes.
Claim 22
Dependent Claim
11 words
The method of claim 1, wherein said sample is blood serum.
Claim 23
Dependent Claim
10 words
The method of claim 1, wherein said sample is urine.
Claim 24
Independent Claim
178 words
A method for the quantitative determination of the pancreatic and salivary α-amylases contained in a sample of a body fluid wherein said fluid initially contains glucose as a contaminant in the quantitative determination comprising the steps of buffering said sample to a suitable pH value, passing said buffered sample through a scavenger stage containing coimmobilized reagents comprising glucose oxidase and catalase wherein said glucose is removed as a contaminant, passing said glucose-free sample through a substrate stage containing an immobilized starch reagent comprising a high amylose content on an alumina support wherein said starch reagent quantitatively reacts with the α-amylases contained in said sample to produce oliqosaccharides, passing said oligosaccharides-containing sample through a glucose-generating stage containing an immobilized reagent comprising glucoamylase to product a glucose reaction product, passing said glucose-containing sample to a detection stage comprising an immobilized glucose oxidase reagent wherein said glucose reaction product is oxidized to generate hydrogen peroxide, determining with detection means the hydrogen peroxide generated and relating the amount of hydrogen peroxide generated to the quantitative amount of α-amylases contained in said sample.
Claim 25
Independent Claim
178 words
A method for the quantitative determination of the pancreatic and salivary α-amylases contained in a sample of a body fluid wherein said fluid initially contains glucose as a contaminant in the quantitative determination comprising the steps of buffering said sample to a suitable pH value, passing said buffered sample through a scavenger stage containing coimmobilized reagents comprising glucose oxidase and catalase wherein said glucose is removed as a contaminant, passing said glucose-free sample through a substrate stage containing an immobilized starch reagent comprising a high amylose content on an alumina support wherein said starch reagent quantitatively reacts with the α-amylases contained in said sample to produce oliqosaccharides, passing said oligosaccharides-containing sample through a glucose-generating stage containing an immobilized reagent comprising glucoamylase to product a glucose reaction product, passing said glucose-containing sample to a detection stage comprising an immobilized glucose oxidase reagent wherein said glucose reaction product is oxidized to generate hydrogen peroxide, determining with detection means the hydrogen peroxide generated and relating the amount of hydrogen peroxide generated to the quantitative amount of α-amylases contained in said sample.
Claim 26
Dependent Claim
18 words
The method of claim 23, wherein said immobilized starch reagent is potato starch cross-linked on said alumina support.
Claim 27
Dependent Claim
23 words
The method of claim 23, wherein said sample is passed through said immobilized reagents of said several stages sequentially in a confined zone.
Claim 28
Dependent Claim
12 words
The method of claim 23, wherein said detection means comprises polarographic means.
Claim 29
Dependent Claim
12 words
The method of claim 23, wherein said detection means comprises spectrographic means.
Claim 30
Independent Claim
174 words
A method for the quantitative determination of the pancreatic and salivary α-amylases contained in an aqueous solution sample containing glucose as a contaminant in the quantitative determination comprising the steps of buffering said sample to a suitable pH value, passing said buffered sample solution through a substrate stage containing an immobilized starch reagent comprising a high amylose content wherein said starch reagent quantitatively reacts with the α-amylases contained in said sample to produce oligosaccharides, passing said oligosaccharides containing sample solution through a scavenger stage containing immobilized reagents wherein said glucose contaminant is removed, mixing said glucose-free, oligosaccharide-containing sample solution with a second buffer solution and passing said buffered sample solution through glucose-generating stage containing immobilized reagents, wherein said reagents produce a glucose reaction product from said oligosaccharides, passing said glucose-containing sample solution to a detection stage werein said glucose reaction product is reacted to generate hydrogen peroxide, passing said hydrogen peroxide containing sample solution through detection means and relating the amount of hydrogen peroxide generated to the quantitative amount of α-amylases in said sample.
Claim 31
Independent Claim
174 words
A method for the quantitative determination of the pancreatic and salivary α-amylases contained in an aqueous solution sample containing glucose as a contaminant in the quantitative determination comprising the steps of buffering said sample to a suitable pH value, passing said buffered sample solution through a substrate stage containing an immobilized starch reagent comprising a high amylose content wherein said starch reagent quantitatively reacts with the α-amylases contained in said sample to produce oligosaccharides, passing said oligosaccharides containing sample solution through a scavenger stage containing immobilized reagents wherein said glucose contaminant is removed, mixing said glucose-free, oligosaccharide-containing sample solution with a second buffer solution and passing said buffered sample solution through glucose-generating stage containing immobilized reagents, wherein said reagents produce a glucose reaction product from said oligosaccharides, passing said glucose-containing sample solution to a detection stage werein said glucose reaction product is reacted to generate hydrogen peroxide, passing said hydrogen peroxide containing sample solution through detection means and relating the amount of hydrogen peroxide generated to the quantitative amount of α-amylases in said sample.
Claim 32
Dependent Claim
18 words
The method of claim 28, wherein said immobilized starch reagent is potato starch cross-linked on a porous support.
Claim 33
Dependent Claim
23 words
The method of claim 28, wherein said sample solution is passed through said scavenger reagents in several stages sequentially in a confined zone.
Claim 34
Dependent Claim
12 words
The method of claim 28, wherein said detection means comprises polarographic means.
Claim 35
Dependent Claim
12 words
The method of claim 28, wherein said detection means comprises sepctrographic means.
Claim 36
Dependent Claim
14 words
The method of claim 28, wherein said scavenger reagents comprise glucose oxidase and catalase.
Claim 37
Dependent Claim
11 words
The method of claim 28, wherein said glucose-generating reagent is glucoamylase.
Claim 38
Dependent Claim
11 words
The method of claim 28, wherein said glucose-generating reagent is maltase.
Claim 39
Dependent Claim
15 words
The method of claim 28, wherein said glucose reaction product is reacted with glucose oxidase.
Claim 40
Dependent Claim
16 words
The method of claim 28, wherein said substrate starch reagent is an amylose starch on alumina.
Claim 41
Dependent Claim
21 words
The method of claim 28, wherein said substrate starch reagent is an amylose starch immobilized and cross-linked on porous acid-activated alumina.
Claim 42
Dependent Claim
12 words
The method of claim 1, wherein said glucose-generating stage comprises immobilized α-amylase.
Claim 43
Dependent Claim
12 words
The method of claim 23, wherein said glucose-generating stage comprises immobilized α-amylase.
Claim 44
Dependent Claim
12 words
The method of claim 28, wherein said glucose-generating stage comprises immobilized α-amylase.
References Cited (Backward Citations)
US4000042A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1982-10-29 | 1983-04-30 | 1983-10-30 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1986-10-29 | 1987-04-30 | 1987-10-30 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1990-10-29 | 1991-04-30 | 1991-10-30 | $7700.00 | Window Closed / Lapsed |